Mosquito gut microbiome is critical for priming mosquito basal immunity. As an effort to understand microbiome in the mosquito gut ecosystem, this project addresses important questions regarding how non-pathogenic bacteria colonize the gut and interact with host immune surveillance to maintain a symbiotic relationship, which in turn provides effective protection against pathogens like malaria. The bacterial cell wall component lipopolysaccharide (LPS) makes a connection of colonization and immunogenicity. We have isolated a predominant gut bacterium Enterobacter sp., from the mosquito gut, and a mutant was generated, in which the gene waaL was disrupted. The LPS of the mutant strain has defects. We will use the wild type and the waal mutant to study the role of LPS in bacterial colonization of the gut and its role in immunogenic potency. We will create an in vivo working model using GFP- or RFP- tagged Enterobacter wild type or mutant to reconstruct the gut population, and then determine its efficiency of colonization. The RNA-seq will be used for analyzing transcriptomes of both Enterobacter and the gut simultaneously, a dual approach to acquire data pertinent to the mutual interactions between the bacteria and host. The impact of the LPS defects on bacterial immunogenicity and host immunity against malaria will also be assessed. The project will generate data to advance our understanding how a symbiotic microbial community is shaped, and how host immune system interacts with co-evolved non-pathogenic microorganisms to maintain symbiostasis in the gut ecosystem. The knowledge will facilitate to develop novel ways to strengthen mosquito anti-malaria immunity to reduce malaria transmission.